基因工程耐热碱性磷酸酯酶的表达与分离纯化  

Expression and purifcation of thermostable alkaline phosphatase produced by genetic engineering

作　　者：朱运良 申成斌[2] 季朝能[3] 李朴 盛小禹[3] 

机构地区：[1]洛阳医专法医学系,洛阳471003 [2]河南省公安厅刑事科学技术研究所,郑州450000 [3]复旦大学遗传学研究所,上海200433

出　　处：《洛阳医专学报》2000年第4期278-280,共3页Journal of Luoyang Medical College

摘　　要：目的　获得可用于探针的非放射性标记的耐热碱性磷酸酯酶。方法　把耐热碱性磷酸酯酶的基因克隆入质粒pJLA5 0 3上 ,在E .coliMph44中表达。表达的酶用聚乙烯亚胺沉淀核酸 ,热变性 ,硫酸铵沉淀 ,层析等方法纯化。结果　经表达纯化后每克细菌可获得 1.5mg的酶 ,酶的纯度为 90 % ,比活为 78.4U/mg。结论　耐热碱性磷酸酯酶可在E .coliMph44中表达 ,表达的酶可用热变性和其它蛋白质纯化方法来纯化。Objective To produce thermostable alkaline phosphatase (FD TAP) which can be used in non radioactive labelling. Methods Cloned into plasmid pJLA503, the enzyme was expressed in E.coli Mph44. The expressed enzyme was purified by means of cell lysis, PEI precipitation, heat denaturation, salting out with ammonium sulfate and CM Sepharose fast flow chromatography. Results Enzyme protein of 90% purity, the specific activity of which was 78.4U/mg,was obtained. The yield per gram of bacteria was about 1.5mg. Conclusions FD TAP could be expressed in E.coli Mph44 with pJLA503 as a vector and the expressed enzyme could be purifed by means of heat denaturation and some protein purification methods.

关 键 词：表达 蛋白纯化 耐热酶 碱性磷酸酯酶 

分 类 号：Q556.1[生物学—生物化学] Q78